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Psychrophilic organisms thriving at near-zero temperatures synthesize cold-adapted enzymes to sustain cell metabolism. These enzymes have overcome the reduced molecular kinetic energy and increased viscosity inherent to their environment and maintained high catalytic rates by development of a diverse range of structural solutions. Most commonly, they are characterized by a high flexibility coupled with an intrinsic structural instability and reduced substrate affinity. However, this paradigm for cold-adaptation is not universal as some cold-active enzymes with high stability and/or high substrate affinity and/or even an unaltered flexibility have been reported, pointing to alternative adaptation strategies. Indeed, cold-adaptation can involve any of a number of a diverse range of structural modifications, or combinations of modifications, depending on the enzyme involved, its function, structure, stability, and evolutionary history. This paper presents the challenges, properties, and adaptation strategies of these enzymes.

Today, the world faces an enormous increase in plastic waste pollution caused by the emergence of the COVID-19 pandemic. Plastic pollution has been already one of the greatest threats to our planet before the Coronavirus outbreak. The disposal of millions of personal protective equipment (PPE) in the form of face masks has significantly contributed to the generation of plastic waste and has exacerbated plastic pollution. In an attempt to mitigate pollution caused by the excess PPE waste, an innovative way was developed in this research to reduce pandemic-generated wastes by using the shredded face mask (SFM) fibers as an additive to hot mix asphalt (HMA) to enhance rutting resistance. Rutting or permanent deformation is one of the major distresses of asphalt pavement. Since the SFM behaves as a semi-liquid between 115.5 and 160 °C, which is in the range of HMA mixing and paving temperature, it can function as a binding agent to glue the aggregates. When the pavement is cooled down to ambient temperature, the hardened SFM can provide stability and stiffness to HMA. Based on the results of this study, the modified mixes exhibited excellent resistance to permanent deformation under the Asphalt Pavement Analyzer (APA), as rutting depth values were reduced from 3.0 mm to 0.93 mm by increasing the SFM content from 0% to 1.5%. From the rutting test results and premature distress mechanism study, the appropriate addition of SFM modifiers could improve the high-temperature properties of HMA that can be used to strengthen high-compression and shearing zones in the pavement structure.

‘What is a nation?’ Ernest Renan famously asked in 1882. The question of what is a sporting nation is perhaps even more complex to answer.Greg Ryan and Geoff Watson's Sport and the New Zealanders i...

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti-Candida activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. In vitro antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of Candida albicans, Candida auris, Candida dubliniensis, and Candida tropicalis. Finally, the ability of macrophage cells to kill Candida isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 mu g/ml of cinnamaldehyde, 256 mu g/ml of citral, and 128 mu g/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (similar to 41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages., This work was supported by the Consejeria de Educacion, Universidades e Investigacion (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza, the Fundacion ONCE Oportunidad al Talento, and the Fondo Social Europeo (to C.M.-A.), and the Federation of European Microbiological Societies (FEMS) by Research and Training grant FEMS-GO-2017-020 (to K.M.-C.).

A prerequisite to the use of any enzyme in any industrial process is an understanding of its activity and stability under process conditions. Glycoside hydrolase family 8 enzymes include many important biotechnological biocatalysts yet little is known of the performance of these with respect to pH. A better understanding of this parameter and its relationship to structure and function in these enzymes will allow for an improved use of these in industry as well as an enhanced ability in their engineering and optimisation for a particular application. An in-depth analysis of the pH induced changes in activity, irreversible inactivation, conformation, stability and solubility of a commercial glycoside hydrolase family 8 xylanase was carried out with the aim of identifying the factors determining the pH dependence of this enzyme. Our study showed that different phenomena play different roles at the various pHs examined. Both reversible and irreversible processes are involved at acidic pHs, with the irreversible processes dominating and being due to protein aggregation and precipitation. At basic pHs, loss of activity is principally due to reversible processes, possibly deprotonation of an essential catalytic residue, but at higher pHs, near the pI of the protein, precipitation again dominates while structure unfolding was discerned at the higher pHs investigated. Such insights demonstrate the complexity of factors involved in the pH dependence of proteins and advances our knowledge on design principles and concepts for engineering proteins. Our results highlight the major role of protein precipitation in activity and stability losses at both low and high pHs but it is proposed that different strategies be used in tailoring the enzyme to overcome this in each case. Indeed the detailed understanding obtained here will allow for a more focused, informed and hence successful tailoring of glycoside hydrolase family 8 proteins for a specific pH and process application., This work was financed by FEDER through POFC-COMPETE and by the Fundação para a Ciência e Tecnologia (FCT) through project EngXyl (EXPL/BBB-BIO/1772/2013 – FCOMP-01-0124-FEDER-041595) as well as through strategic funding via UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and by the ERDF through Compete 2020 – POCI. G.F. is supported by the Belgian program of Interuniversity Attraction Poles (iPros P7/44). M.B. acknowledges the FCT for grant PD/BD/113810/2015 within the Doctoral Program in Applied and Environmental Microbiology. T.C. is supported by the FCT, the European Social Fund, the Programa Operacional Potencial Humano and the Investigador FCT Programme (IF/01635/2014)., info:eu-repo/semantics/publishedVersion

Microorganisms in cold ecosystems play a key ecological role in their natural habitats. Since these ecosystems are especially sensitive to climate changes, as indicated by the worldwide retreat of glaciers and ice sheets as well as permafrost thawing, an understanding of the role and potential of microbial life in these habitats has become crucial. Emerging technologies have added significantly to our knowledge of abundance, functional activity, and lifestyles of microbial communities in cold environments. The current knowledge of microbial ecology in glacial habitats and permafrost, the most studied habitats of the cryosphere, is reported in this review., Open access funding provided by University of Innsbruck and Medical University of Innsbruck. T.C. is supported by the Fundacao para a Ciencia e a Tecnologia (FCT), the European Social Fund, the Programa Operacional Potencial Humano, and the Investigador FCT Programme (IF/01635/2014)., info:eu-repo/semantics/publishedVersion

Cold-adapted microorganisms inhabiting permanently low-temperature environments were initially just a biological curiosity but have emerged as rich sources of numerous valuable tools for application in a broad spectrum of innovative technologies. To overcome the multiple challenges inherent to life in their cold habitats, these microorganisms have developed a diverse array of highly sophisticated synergistic adaptations at all levels within their cells: from cell envelope and enzyme adaptation, to cryoprotectant and chaperone production, and novel metabolic capabilities. Basic research has provided valuable insights into how these microorganisms can thrive in their challenging habitat conditions and into the mechanisms of action of the various adaptive features employed, and such insights have served as a foundation for the knowledge-based development of numerous novel biotechnological tools. In this review, we describe the current knowledge of the adaptation strategies of cold-adapted microorganisms and the biotechnological perspectives and commercial tools emerging from this knowledge. Adaptive features and, where possible, applications, in relation to membrane fatty acids, membrane pigments, the cell wall peptidoglycan layer, the lipopolysaccharide component of the outer cell membrane, compatible solutes, antifreeze and ice-nucleating proteins, extracellular polymeric substances, biosurfactants, chaperones, storage materials such as polyhydroxyalkanoates and cyanophycins and metabolic adjustments are presented and discussed., T.C. is supported by the FCT, the ESF, POPH, andthe Investigador FCT Programme (IF/01635/2014). Funding by the ERDF is through project EcoAgriFood (NORTE-01-0145-FEDER-000009) via the North Portugal Regional Operational Programme(NORTE 2020) under the PORTUGAL 2020 Partnership Agreement.FCT funding was through the project EngXyl (EXPL/BBB-BIO/1772/2013-FCOMP-01-0124-FEDER-041595) and the strategic programme UID/BIA/04050/2019 at the CBMA., info:eu-repo/semantics/publishedVersion

Lignocellulosic hydrolysates remain one of the most abundantly used substrates for the sustainable production of second generation fuels and chemicals with Saccharomyces cerevisiae. Nevertheless, fermentation inhibitors such as acetic acid, furfural and hydroxymethylfurfural are formed during the process and can lead to slow or stuck fermentations and/or act as genotoxic agents leading to production strain genetic instability. We have developed a novel dominant deletion (DEL) cassette assay for quantification of DNA damage in both wild-type and industrial yeast strains. Using this assay, the ethanol production strain S. cerevisiae PE-2 was shown to be more resistant to hydrogen peroxide and furfural than the laboratory DEL strain RS112. Indeed, the PE-2 strain also showed a lower tendency for recombination, consistent with a more efficient DNA protection. The dominant DEL assay presented herein should prove to be a useful tool in the selection of robust yeast strains and process conditions for second generation feedstock fermentations., This work was supported by the Fundação para a Ciência e a Tecnologia Portugal (FCT) through Project MycoFat PTDC/AAC-AMB/120940/2010. CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020—Programa Operacional Competitividade e Internacionalizacao (POCI). This work is supported by: European Investment Funds by FEDER/COMPETE/POCI— Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013. T. Collins thanks the FCT for support through the Investigador FCT Programme (IF/01635/2014)., info:eu-repo/semantics/publishedVersion

A qualification methodology for advanced rotary shouldered threaded connections is presented. It covers physical tests of connection prototypes and virtual tests with modeling and simulation techniques. The methodology had been applied to qualify a fatigue-resistant threaded connection design that has recently been released for field testing. The qualification process consists of in-lab makeup and breakout tests, on-rig makeup and breakout tests, sealability tests, fatigue tests, torsional yield limit tests, and tensile capacity tests. During the development of the design, modeling and simulation techniques were extensively used to optimize the design prior to physical prototyping and testing. Some of the qualification tests, such as torsional yield limit tests and tensile capacity tests, were carried out by using advanced modeling and simulation techniques. Because fatigue tests under nominal loads take a long time to complete, accelerated fatigue tests under calculated overloads were conducted to assess whether the design would meet the fatigue life requirements with low risk. After positive results were obtained from the accelerated fatigue tests, nominal loads required by the product specifications were then applied to complete the qualification fatigue tests. It was found from the experimental results that the predicted performance of the connections, such as fatigue life, critical sealing pressure, and breakout torque, matched with the physical test results well. Despite the fact that numerous rotary shouldered threaded connection designs have been developed in the oil and gas industry, there has been no formally defined qualification procedure for such designs. The objective of this paper is to introduce, for the first time, a systematic and complete qualification procedure for rotary shouldered threaded connections.

Free standing films of a genetically engineered silk-elastin-like protein (SELP) were prepared using water and formic acid as solvents. Exposure to methanol-saturated air promoted the formation of aggregated β-strands rendering aqueous insolubility and improved the mechanical properties leading to a 10-fold increase in strain-to-failure. The films were optically clear with resistivity values similar to natural rubber and thermally stable up to 180 °C. Addition of glycerol showed to enhance the flexibility of SELP/glycerol films by interacting with SELP molecules through hydrogen bonding, interpenetrating between the polymer chains and granting more conformational freedom. This detailed characterization provides cues for future and unique applications using SELP based biopolymers.

N/A The origins of the modern football codes have attracted considerable attention from historians of sport over the past two decades, resulting in a vigorous debate between the self-described ‘revisionists’, led by Adrian Harvey, and the followers of Eric Dunning, dubbed by their opponents as the ‘orthodox’ view. However, this article argues that both sides commit the same methodological errors: an overestimation of the importance of written rules, an ahistoric view of culture and continuity, and a tendency to view the past through the lens of the present. By re-examining the historical record of early forms of football and presenting a broader contextual perspective for the emergence of the football codes in the 1840 to 1880 period, the article aims to address some of the key historiographical issues that confront historians of sport today.

Background: Applying very high gravity (VHG) fermentation conditions to the sugarcane juice (SCJ) bioethanol industry would improve its environmental and economic sustainability without the need for major infrastructure changes or investments. It could enable a decrease in the consumption of biological and natural resources (cane/ land, water and energy) while maintaining acceptable production parameters. The present study attempts to demonstrate and characterise an efective industrially relevant SCJ-VHG fermentation process. Results: An industry-like SCJ-VHG bioethanol production process with 30 and 35 °Bx broth was employed to investigate the efects of both the yeast strain used and nitrogen source supplementation on process yield, process productivity, biomass viability, glycerol concentration and retention-associated gene expression. Process performance was shown to be variably afected by the diferent process conditions investigated. Highest process efciency, with a 17% (w/v) ethanol yield and only 0.2% (w/v) sugar remaining unfermented, was observed with the Saccharomyces cerevisiae industrial strain CAT-1 in 30 °Bx broth with urea supplementation. In addition, efcient retention of glycerol by the yeast strain was identifed as a requisite for better fermentation and was consistent with a higher expression of glycerol permease STL1 and channel FPS1. Urea was shown to promote the deregulation of STL1 expression, overcoming glucose repression. The consistency between Fps1-mediated ethanol secretion and ethanol in the extracellular media reinforces previous suggestions that ethanol might exit the cell through the Fps1 channel. Conclusions: This work brings solid evidence in favour of the utilisation of VHG conditions in SCJ fermentations, bringing it a step closer to industrial application. SCJ concentrated up to 30 °Bx maintains industrially relevant ethanol production yield and productivity, provided the broth is supplemented with a suitable nitrogen source and an appropriate industrial bioethanol-producing yeast strain is used. In addition, the work contributes to a better understanding of the VHG-SCJ process and the variable efects of process parameters on process efciency and yeast strain response. Keywords: Biofuel, Bioethanol, Sugarcane, Saccharomyces cerevisiae, CAT-1, Very high gravity, Process optimisation, Process sustainability, B. Monteiro was supported by the Ph.D. Grant 2011/12185-0 from the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP. P. Ferraz and M. Barroca are supported by the Doctoral Programme in Applied and Environmental Microbiology (DP-AEM) and the FCT by Ph.D. Grants PD/ BD/113814/2015 and PD/BD/113810/2015, respectively. T. Collins thanks the FCT for support through the Investigador FCT Programme (IF/01635/2014). T. Collins and C. Lucas are supported by the strategic programme UID/ BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and the ERDF through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). P. Ferraz, T. Collins and C. Lucas were further funded by the project EcoAgriFood (NORTE-01- 0145-FEDER-000009), supported by the Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins., (undefined), info:eu-repo/semantics/publishedVersion

Introduction (Tony Collins and John Nauright) Part 1: Professional Rugby On and Off the Field 1. Professional Rugby and Irish Society 1995-2015 (Liam O'Callaghan) 2. Cows in the Heartland: New Zealand Rugby and Rural Change in the Professional Era (Greg Ryan) 3. The World Comes to One Country: Migration, Cultures and Professional Rugby in France (Philip Dine) 4. Cultural Diversity in Action: Developing and Engaging Effective Responses within Rugby Union in Australia (Jioji Ravulo) 5. The Maori All Blacks and Ethnicity in Aotearoa/New Zealand in the Professional Era (Farah Palmer) 6. The Impact of the Professional Era on Pacific Islands Rugby (Robert Dewey) 7. The Globalisation of Rugby 7s: From Novelty to the Olympic Games (Jeremy Stewart and Marc Keech) 8. The Rugby World Cup As Global Mega Event (Kamilla Swart) Part 2: Rugby Cultures and Representation in the Professional Era 9. Making Men in the 21st Century: Metrosexuality and Bromance in Contemporary Rugby (Adam White and Eric Anderson) 10. The Road From Wigan Pier: Professional Rugby and the Changing Dynamics Between Wales and England (John Harris) 11. 'When Jerseys Speak': Contested Heritage and South African Rugby (Marizanne Grundlingh) 12. Performativity, Identities and Rugby From Field to Stage in the New South Africa (Carla Lever) 13. Dressed for Success: Historicizing Nelson Mandela's Involvement in the 1995 Rugby World Cup (Albert Grundlingh)